While much has been learned about the interactions between Human Immunodeficiency Virus type 1(HIV-1) and the human immune system, many questions remain unanswered, most importantly how best to stimulate and sustain effective anti-HIV-1 immunity with a preventive or therapeutic vaccine. We believe that the careful and detailed interrogation of the interaction of HIV-1 with immune cells in the lymph node will be critical for improving our ability to stimulate anti-HIV-1 immunity. The powerful new technology of multiphoton intravital microscopy (MV-IVM) has allowed for the visualization of immune cell migration and cell-cell interaction in lymph nodes, but current MP-IVM technology can only be applied to small animals, such as rodents. There has been long-standing interest in developing a small animal model of HIV-1 infection, and the advent of MP-IVM gives additional impetus to achieve this goal. Recently, we and others have developed a markedly improved humanized mouse model of HIV-1 by transplanting human CD34+ stem cells and autologous human thymic grafts into immunodeficient mice. These mice generate robust anti-HIV- 1 cellular and humoral immune responses, and achieve repopulation of their lymph nodes with human immune cells adequate to support MP-IVM studies. Using MP-IVM, we have demonstrated, for the first time, that adoptively transferred human T cells can home to humanized mouse lymph nodes from the blood and migrate normally once in the lymph node. In Aim 1 of this project, we will perform, for the first time, a thorough analysis of the migratory behavior of human T cells and dendritic cells in a humanized mouse. This unique study will allow a more complete understanding of this potentially important model of the human immune system in action, in real time. Then in Aim 2, we will take advantage of this improved humanized mouse model to study questions regarding the biology of HIV-1 not readily approachable through human studies. Using fluorescently labeled virus, we will determine how HIV-1 is delivered to the lymph node either as a free virus or a virus associated with dendritic cells or T cells. We will then determine the anatomy of HIV-1 spread within the LN over time using an HIV-1 mutant inducing the expression of GFP in infected cells. In addition to addressing these questions of viral transport and cell-cell interactions, we will be able to use our improved model of HIV-1 infection to address important questions of immune cell function in the context of HIV-1 infection. As a dramatic example of the usefulness of our mouse model of HIV-1 infection, we have found that similar to humans infected with HIV-1, CD4+ and CD8+ T cells in our humanized mice dramatically increase their cell surface expression of PD-1 following HIV-1 infection. PD-1 negatively regulates T cell activity, is highly expressed on HIV-1-specific T cells and is associated with T cell exhaustion and predictors of HIV-1 progression. In Aim 3, we will use our humanized mouse model of HIV-1 to determine if blockade of the PD-1 pathway will reinvigorate the cellular and humoral immune response to HIV-1 and lead to better control of HIV-1 replication. The project will benefit from and synergize with the expertise of the other investigators in this PPG focused on viral transport in lymph nodes, the biology of the PD-1 family, and the humoral immune response to pathogens, as well as the BL2+-contained MP-IVM core.